In the process of creating a species cross or genus cross of fish or other aquatic animals, embryogenesis begins with the fertilization of an egg. However, most hybrids thereby created will be finally dead, and—those hybrids which remain alive will be sterile due to pairing failure, which results from the nonhomogenity of their chromosomes. Crossing between allied species can make the pairing successful and provide them with fertility. However, the next generation would suffer from genetic separation, so that their qualities superior to those of the parents (i.e. the hybrid vigor) will be gradually lost, generation by generation.
FIGS. 11 and 12 schematically show genomic combinations in the creation of the aforementioned hybrids and in the crossing among the members of the first filial generation. It should be noted that the Claims and Description sections and the drawings of the present patent application use the following notations: Genomes (or chromosomes) of different species (A and B) are generally referred to as “A” and “B”, respectively. Sex chromosomes are denoted by “X” and “Y.” If a genome of species A or B needs to have its sex chromosome explicitly indicated, the chromosome of species A is referred to “AX” or “AY” and the chromosome of species B as “BX” or “BY.” In the case where an individual of a fish having genomes AA has laid an egg, the egg retains the second polar body immediately before fertilization. Therefore, this egg is referred to as AA even if it is a reductive egg. The egg will be a real reductive egg (A) for the first time after it has released the second polar body subsequent to its fertilization. Accordingly, a reductive egg is regarded as “A” even if it is an unfertilized egg, except when the second polar body is retained.
In FIG. 11, the chromosome constitution of the first filial generation is either AXBX or AXBY. These combinations may be lethal or sterile, as explained earlier, depending on the constitution of genome A on the maternal side and genome B of the paternal side. Some hybrids may keep living and be fertilized, but they will undergo genetic separation, as shown in FIG. 12. For example, let the chromosomes constituting the genomes A and B of an F1 hybrid (AB) denoted by a1, a2 and a3 and b1, b2 and b3, respectively. Provided that the meiotic division normally takes place, the resultant gamete will have eight possible chromosome combinations, including a1 or b1 as the first chromosome, a2 or b2 as the second chromosome, and a3 or b3 as the third chromosome. Therefore, the F2 hybrids will have 64 possible combinations, some of which have two chromosomes of the same kind combined for each of the first through third chromosomes, such as a1a1 or a3a3. In the most extreme case, the resultant combination can consist of only the chromosomes of species A: a1a1a2a2a3a3. Thus, the qualities of the hybrids are separated into species A or B at each chromosome (or at each gene, if a crossover is taken into account).
It is generally believed that the lethality of the species hybrid or genus hybrid can be avoided by polyploidizing the chromosomes, although its effect depends on the combination of the parent species. Polyploidization, particularly the polyploidization resulting from the prevention of somatic division, is practically used for a variety of plants but barely used for animals.
With respect to the techniques for creating an autotriploid in the field of aquatic animals, particularly fish, there are two patent documents disclosing such techniques relating to sweetfish and flatfish (Unexamined Japanese Patent Application Publication Nos. H10-150883 and H10-327706. The former is called “Patent Document 1” and the latter “Patent Document 2” hereinafter). Concerning the creation of an allotriploid or allotetraploid (which is called the “amphidiploid” hereinafter), there is a report on the successful creation of an allotriploid of salmon (for example refer to Nippon Suisan Gakkai ed. 1989. Suisan Zouyoushoku To Senshokutai Sousa. Tokyo: Kouseisha-kouseikaku. pp. 87-92. This document is called “Non-Patent Document 1” hereinafter). However, concerning amphidiploids, the techniques proposed thus far are only theoretical, general ones except for some unusual cases (see Non-Patent Document 1). Neither has there been any report on the existence of an amphidiploid of a dioecious aquatic animal in the natural world, as opposed to plants, which are capable of self-fertilization.
With reference to FIG. 13, a technique of creating an allotriploid and an amphidiploid of a fish is outlined. FIG. 13 and the technique described below are based on FIG. 8-2 and its description in Non-Patent Document 1.
To create an allotriploid, an egg of species A (an egg in the middle of the second maturation division, with chromosomes AXAX) is fertilized with a sperm of species B (chromosome BX or BY) (the first step in FIG. 13). Then, the egg is subjected to a temperature or pressure to suppress the release of the second polar body (the second step). With the second polar body thus retained, the egg now has a triploid chromosome constitution (AAB) (the third through fifth steps). Therefore, the fish finally obtained will be an allotriploid (AXAXBX or AXAXBY).
To create an allotetraploid (i.e. amphidiploid), an egg of species A is fertilized with a sperm of species B. Then, without suppressing the release of the second polar body, the first cleavage is prevented or suppressed in the fifth step (it should be noted that, according to a study of the present inventor, what is actually suppressed hereby is not the first cleavage but the second one, as will be explained later). The egg thus obtained has a tetraploid chromosome constitution (AABB). Therefore, the fish finally obtained will be an amphidiploid.
In a specific example of the creation of an amphidiploid, Oryzias luzonensis was crossed with Oryzias curvino (Nippon Suisan Gakkaishi, 1993, 59: 373. This document is called “Non-Patent Document 2” hereinafter). This technique is not a mere replication of the method disclosed in Non-Patent Document 1; it further includes the step of inseminating a nonreductive egg AXBX (AXAXBXBX before the release of the polar body) of a cross breed with a sperm genetically inactivated by an irradiation of gamma ray, ultraviolet ray, X-ray or similar radiation. This treatment prevents the second maturation cleavage of the inseminated egg; thereby causing the egg to be an amphidiploid.
The amphidiploid thus created has AXAXBXBX chromosomes. The sister chromosomes function like homologous chromosomes, so that the fertility is restored. As long as the gynogenesis using the eggs produced by this amphidiploid is continued, the progeny individuals will be genetically identical and no male will appear.
The allotriploid mentioned earlier can solve the problem of lethality of the species hybrid or genus hybrid. However, since its chromosome constitution is AXAXBX or AXAXBY, the allotriploid will usually be sterile due to pairing failure of the chromosomes during the meiotic division. Therefore, every time a species hybrid of a specific kind is demanded, it is necessary to repeat the previously described treatment. This means that this technique of creating an allotriploid does not enable the progenies to be produced by natural crossbreeding. This fact restricts the application of this technique in the field of aquaculture and propagation.
Amphidiploids of aquatic animals are said to be theoretically possible. However, there has been only one successful case, as explained earlier. Since that successful case is a kind of gynogenesis, the individuals thereby created are all female (AXAXBXBX). The amphidiploid created in the theoretical example shown in FIG. 13 will be either a female having AXAXBXBX chromosomes or a male having AXAXBYBY chromosomes. However, there has not been any report on a successful creation of an amphidiploid by this theoretical technique. Thus, currently, there is no evidence that a male amphidiploid actually exists. Moreover, the theory predicts only the possibility of AXAXBYBY.
For the reasons described above, amphidiploids are technically more difficult to use in the aquaculture and propagation of aquatic animals than allotriploids.
Thus, the present invention intends to provide an amphidiploid aquatic animal and a method of breeding such an animal, which have the following features: the hybrids are free from lethality and sterility; the hybrid vigor is maintained also in the progeny; the subsequent generations can be created by normal crossing in a stable manner; and the inbreeding depression due to relative mating is totally eliminated.